We have adapted an established Ampliseq microhaplotype panel for nanopore sequencing with the Oxford Nanopore Technologies (ONT) system, as a cost-effective and highly scalable solution for forensic genetics applications. For this purpose, we designed a protocol combining direct PCR amplification from unextracted DNA with ONT library construction and sequencing using the MinION device and workflow. The analysis of reference samples at input amounts of 5–10 ng of DNA demonstrates stable coverage patterns, allele balance, and strand bias, reaching profile completeness and concordance rates of ∼95%. Similar levels were achieved when using direct-PCR from blood, buccal and semen swabs. Dilution series results indicate sensitivity is maintained down to 250 pg of input DNA, and informative profiles are produced down to 62.5 pg. Finally, we demonstrated the forensic utility of the nanopore workflow by analyzing two third degree pedigrees that showed low likelihood ratio values after the analysis of an extended panel of 38 STRs, achieving likelihood ratios 2–3 orders of magnitude higher when testing with the MinION–based haplotype data.

我们采用牛津纳米孔技术 (ONT) 系统对已建立的 Ampliseq 微单倍型面板进行纳米孔测序，作为法医遗传学应用的经济高效且高度可扩展的解决方案。为此，我们设计了一种方案，将未提取 DNA 的直接 PCR 扩增与使用 MinION 设备和工作流程进行 ONT 文库构建和测序相结合。对输入量为 5-10 ng DNA 的参考样本进行分析，显示出稳定的覆盖模式、等位基因平衡和链偏倚，达到了约 95% 的谱完整性和一致性。当使用血液、口腔和精液拭子的直接 PCR 时，也达到了类似的水平。稀释系列结果表明，输入 DNA 的浓度低至 250 pg 时仍能保持灵敏度，低至 62.5 pg 时仍可产生信息丰富的谱。最后，我们通过分析两个三级谱系证明了纳米孔工作流程的法医实用性，这两个谱系在分析 38 个 STR 的扩展面板后显示出较低的似然比值，在使用 MinION 进行测试时，似然比提高了 2-3 个数量级–基于单倍型数据。